2008 — 2012 |
Tsang, Michael Waikok |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Delineating the Role of Fgf Signaling and Vertebrate Heart Development @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Congenital heart defects are one of the most common genetic anomalies, and is the leading cause of deaths in infants. The biology of how cardiac progenitor cells become the first functional organ in the embryo is therefore an important process to resolve. The cardiac progenitor cells are initially specificed very early in development as two populations of cells within the embryo that later migrate and to meet at the midline and form the heart tube. Eventually through a process known as morphogenesis this population of cells evolve to build a functional heart that provides the pump for nutritional and waste exchange in the embryo. This proposal aims to elucidate the role for the Fibroblast Growth Factor signaling (FGF) and (ETS) transcription factors in heart formation. FGFs have been implicated to play an important role in heart formation and mutations in components of this signaling pathway that alter cellular communication during embryogenesis are the cause of human genetic disease, that often includes cardiac defects. We will test the hypothesis that FGFs and ETS factors are required during the earliest phases of cardiac progenitor specification (Aim 1). More important we will determine the mechanism of gene regulation by FGFs and Ets factors (Aim 2). Further, we have identified a small molecule that hyperactives FGF signaling in the embryo and future studies will determine its affects on cardiac development (Aim 3). Understanding how FGFs can alter cell fate and the genes that they control to achieve this is a fundamental question of how cells are molded into organs. This proposal will combine the embryological and genetic features of the zebrafish embryo to answer these questions. PUBLIC HEALTH RELEVANCE. The manual for heart formation is written in as a set of detail instructions encoded in the DNA. How these instructions are read and implemented by cells that eventually become a functional beating organ is the focus of this proposal. Specifically the goal is to understand how a family of ETS transcriptional factors can direct cardiac development in the zebrafish. Another goal related to heart development is the idea that chemical compounds can be used to influence heart growth and differentiation. To reach this goal, we have developed a zebrafish biosensor that can report on signaling activity and have identified a small molecule that can expand cardiac progenitors during development. Understanding how this molecule acts to increase heart tissue is an important step towards developing potential treatment for cardiac damage caused by heart disease.
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0.958 |
2012 — 2016 |
Hukriede, Neil A. [⬀] Tsang, Michael Waikok Vogt, Andreas |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Small Molecule Screens to Identify Probes For Studies of Repair and Regeneration @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): The zebrafish is an important genetic model for studying embryonic patterning and organogenesis. Although genetic tools are available to probe gene function and signaling pathways during embryonic development, their utility is limited with temporally tightly controlled processes or with genes whose perturbation results in embryo lethality. Small molecule probes can overcome these obstacles due to their rapid and reversible actions, thereby enhancing genetic studies and offering a unique opportunity to uncover the roles of signaling pathways in larval and adult physiology. Currently, methods to study gene function in adult zebrafish involves the generation of transgenic heat shock driver lines, the use of binary gene activation such as the Gal4-UAS system, or the use of genetic recombination such as Cre recombinase to activate gene expression. Sophisticated tissue specific gene knockouts are not currently feasible in zebrafish, thus limiting the study of signaling pathways to early development, when gene products can be knocked-down with antisense oligonucleotides. The objective of this proposal is to identify novel small molecule modulators of the FGF and TGFß pathways as tools to dissect the role of these signaling pathways in zebrafish larval and adult repair and regeneration. The FGF and TGFß signaling pathways are critical in regeneration, repair, and wound healing but their exploitation as potential pharmacological targets awaits elucidation of their precise molecular mechanisms during these events. Small molecules that hyper-activate these pathways would be useful tools to study the roles of these pathways and represent starting points for the development of novel regenerative therapies. Ultimately, we will provide the zebrafish community with a unique set of tools to study later stages of development and adult zebrafish models of disease. These studies will provide validated probes for enhancing FGF and TGFß signaling with defined specificity and in vivo activity in models of tissue repair and regeneration. The proposed work is divided into three specific aims, which take advantage of the complementary expertise of investigators on this multi-PI proposal. Aim 1: We will identify compounds that activate the FGF signaling pathway. Aim 2: We will identify compounds that activate the TGFß signaling pathway. Aim 3: We will test the efficacy of the new compounds in regeneration models that are commonly used in our laboratories.
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0.958 |
2014 — 2017 |
Chennubhotla, Srinivas Chakra (co-PI) [⬀] Lo, Cecilia W. Tsang, Michael Waikok |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Assaying Heterotaxy Patient Genes in Cilia Motility and Left-Right Patterning @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Heterotaxy, a birth defect involving randomized left-right patterning of visceral organs, is frequently associated with complex congenital heart disease (CHD), a reflection of the importance of left-right patterning in formation of asymmetries in the four-chamber heart. Heterotaxy (HTX) patients have unexplained higher morbidity and mortality, often with increased postsurgical respiratory complications. This may reflect the common requirement for motile cilia, both in embryonic left-right patterning and also mucus clearance in the airway. We recently showed 42% of HTX patients with CHD (HTX/CHD) have airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD), a recessive disorder associated with laterality defects and sinopulmonary disease due to mucus clearance defects caused by immotile/dyskinetic cilia in the airway. Significantly, exome sequencing showed HTX patients with CD (HTX/CD) are enriched for novel/rare coding variants (RCV) in genes known to cause PCD and other cilia related genes. In this application, we will functionally assay 53 cilia candidate genes identified in 39 HTX/CD patients by exome sequencing analysis. We will assess the effects of gene knockdown on airway cilia motility using a novel assay with reciliating human airway epithelial cells. To assay gene function required for left-right patternin, antisense MO knockdown in zebrafish embryos will be carried out to examine heart and gut looping. Genes shown to disrupt airway cilia motility and cause HTX after knockdown will be further tested to determine whether the RCVs are pathogenic. Specifically we will examine whether expression of the RCV can rescue the HTX phenotype elicited by MO gene knockdown in the zebrafish embryo. Given all of the RCVs identified in HTX/CD patients were heterozygous, we hypothesize a multigenic model of disease, which will be tested by examining the phenotypes of double heterozygous mouse and zebrafish mutants with two-gene combinations observed in the HTX-CD patients. We will examine for evidence of digenic interactions by assaying motile cilia function and visceral organ situs in the double heterozygous mutants. These experiments will interrogate 8 digenic combinations that make use of 9 novel mouse mutants recovered from our ongoing mouse mutagenesis screen, and 7 other digenic combinations in zebrafish using existing mutant lines and de novo production of 4 zebrafish knockout lines by TALENs gene disruption. Finally, to establish genotype-phenotype correlation in ciliary motion defects, we will develop software for quantitative classification of ciliary motin defects using a computational approach with computer vision and machine learning algorithms for visual pattern recognition. Using this software, we will determine whether different RCVs are associated with different ciliary motion defects. This will provide insights into structure-functio relationships in the regulation of cilia motility. This software, to be made available as an online tool, will have translational potential for clinical evaluation of patient airway ciliary motion daa. Together, these studies will establish functional assays and software that can elucidate the genetic etiology of CHD/HTX.
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0.958 |
2018 — 2021 |
Lo, Cecilia W. Tsang, Michael Waikok |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Lv Hypoplasia in Hypoplastic Left Heart Syndrome @ University of Pittsburgh At Pittsburgh
Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect (CHD) characterized by a small left ventricle (LV) and hypoplastic aorta and aortic/mitral valves. A genetic etiology for HLHS is strongly indicated by high recurrence risk, but the genetic underpinning for HLHS is poorly understood. Clinical studies suggest HLHS is multigenic and genetically heterogeneous. Insights into the genetics of HLHS has come from our recent recovery of the first mouse models of HLHS from a large-scale mouse mutagenesis screen. From 8 independent HLHS mouse lines recovered, 330 mutations were identified, with no genes shared in common between the 8 lines. These findings indicate HLHS is profoundly genetically heterogeneous, consistent with the human studies. Detailed analysis of one mutant mouse line, Ohia, showed HLSH is elicited by mutations in two genes: Sap130, a Sin3a associated protein in the chromatin modifying histone deacetylase complex (HDAC), and Pcdha9, a protocadherin mediating cell-cell adhesion. The LV hypoplasia was shown to be elicited by the Sap130 mutation, a finding confirmed with replication of a small ventricle phenotype in a CRISPR generated sap130a zebrafish mutant. The LV hypoplasia was associated with a cardiomyocyte cell proliferation defect and cardiomyocyte cell cycle arrest. In this study, we will investigate the cellular and molecular mechanisms and genetic interactions driving the LV hypoplasia in HLHS, leveraging the unique strengths of the zebrafish and mouse models. In Aim 1, we will employ lineage tracing studies in zebrafish and experiments with Cre deletion of Sap130 in mice to test the hypothesis that Sap130 functions in a cell autonomous manner to regulate ventricular/LV growth. These studies will delineate the cellular context in which Sap130 regulates LV growth. In Aim 2, we will investigate the hypothesis that the hypomorphic Sap130Ohia mutation causes LV hypoplasia via target genes that regulate cardiomyocyte cell cycle and cell proliferation. These studies will focus on Meis1, a Sap130 target gene, also known to regulate cardiomyocyte cell cycle and postnatal cell cycle arrest. In parallel, additional candidate genes identified via Sap130 ChIP-seq and RNA-seq analysis will be assessed for their role in LV hypoplasia with production and analysis of CRISPR targeted embryos and mice. In Aim 3, we will probe the interaction of chromatin modifiers with the Ras/MAPK signaling pathway in the pathogenesis of HLHS using antisense morpholino gene knockdown in zebrafish with a sensitized genetic background. Positive genetic interactions will be validated using mutant or CRISPR targeted zebrafish or mice. This study is motivated by the unexpected recovery of mutations in chromatin modifiers and Ras/MAPK pathway components in all 8 HLHS mouse lines, suggesting chromatin modifiers in combination with dysregulated Ras/MAPK signaling may contribute to the LV hypoplasia and complex genetics of HLHS. Together these studies will help to elucidate the cellular and molecular mechanisms driving the ventricular hypoplasia in HLHS, findings that may yield new therapeutic targets for fetal intervention to recover LV growth. !
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0.958 |
2020 |
Lo, Cecilia W. Tsang, Michael Waikok |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Lv Hypoplasia in Hypoplastic Left Heart Syndrome Supplement @ University of Pittsburgh At Pittsburgh
Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect (CHD) characterized by a small left ventricle (LV) and hypoplastic aorta and aortic/mitral valves. A genetic etiology for HLHS is strongly indicated by high recurrence risk, but the genetic underpinning for HLHS is poorly understood. Clinical studies suggest HLHS is multigenic and genetically heterogeneous. Insights into the genetics of HLHS has come from our recent recovery of the first mouse models of HLHS from a large-scale mouse mutagenesis screen. From 8 independent HLHS mouse lines recovered, 330 mutations were identified, with no genes shared in common between the 8 lines. These findings indicate HLHS is profoundly genetically heterogeneous, consistent with the human studies. Detailed analysis of one mutant mouse line, Ohia, showed HLSH is elicited by mutations in two genes: Sap130, a Sin3a associated protein in the chromatin modifying histone deacetylase complex (HDAC), and Pcdha9, a protocadherin mediating cell-cell adhesion. The LV hypoplasia was shown to be elicited by the Sap130 mutation, a finding confirmed with replication of a small ventricle phenotype in a CRISPR generated sap130a zebrafish mutant. The LV hypoplasia was associated with a cardiomyocyte cell proliferation defect and cardiomyocyte cell cycle arrest. In this study, we will investigate the cellular and molecular mechanisms and genetic interactions driving the LV hypoplasia in HLHS, leveraging the unique strengths of the zebrafish and mouse models. In Aim 1, we will employ lineage tracing studies in zebrafish and experiments with Cre deletion of Sap130 in mice to test the hypothesis that Sap130 functions in a cell autonomous manner to regulate ventricular/LV growth. These studies will delineate the cellular context in which Sap130 regulates LV growth. In Aim 2, we will investigate the hypothesis that the hypomorphic Sap130Ohia mutation causes LV hypoplasia via target genes that regulate cardiomyocyte cell cycle and cell proliferation. These studies will focus on Meis1, a Sap130 target gene, also known to regulate cardiomyocyte cell cycle and postnatal cell cycle arrest. In parallel, additional candidate genes identified via Sap130 ChIP-seq and RNA-seq analysis will be assessed for their role in LV hypoplasia with production and analysis of CRISPR targeted embryos and mice. In Aim 3, we will probe the interaction of chromatin modifiers with the Ras/MAPK signaling pathway in the pathogenesis of HLHS using antisense morpholino gene knockdown in zebrafish with a sensitized genetic background. Positive genetic interactions will be validated using mutant or CRISPR targeted zebrafish or mice. This study is motivated by the unexpected recovery of mutations in chromatin modifiers and Ras/MAPK pathway components in all 8 HLHS mouse lines, suggesting chromatin modifiers in combination with dysregulated Ras/MAPK signaling may contribute to the LV hypoplasia and complex genetics of HLHS. Together these studies will help to elucidate the cellular and molecular mechanisms driving the ventricular hypoplasia in HLHS, findings that may yield new therapeutic targets for fetal intervention to recover LV growth. !
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0.958 |